Method and particle analyzer for determining a broad particle size distribution

ABSTRACT

A method and a particle analyzer are provided for determining a particle size distribution of a liquid sample including particles of a lower size range, particles of an intermediate size range, and particles of an upper size range. A dark-field image frame is captured in which the particles of the lower size range and the particles of the intermediate size range are resolved, and a bright-field image frame is captured in which the particles of the intermediate size range and the particles of the upper size range are resolved. Absolute sizes of the particles of the intermediate size range and the particles of the upper size range are determined from the bright-field image frame. Calibrated sizes of the particles of the lower size range are determined from the dark-field image frame by using the particles of the intermediate size range as internal calibration standards.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods and particle analyzers fordetermining particle size distributions of liquid samples. Moreparticularly, the present invention relates to methods and particleanalyzers for determining particle size distributions by usingbright-field and/or dark-field imaging.

BACKGROUND OF THE INVENTION

The determination of particle size distributions of liquid samples isnecessary in pharmaceutical, life-science, and environmental-scienceapplications, among others. The liquid samples may include dispersedparticles of sizes ranging from less than 0.1 μm to 1000 μm. Generally,different types of particle analyzer are used to determine the sizes ofparticles of different size ranges.

Bright-field imaging particle analyzers may be used to determine thesizes of particles larger than about 0.5 μm in a liquid sample. Forexample, micro-flow imaging (MFI) particle analyzers are described inU.S. Pat. No. 7,064,826 to Rabinski, et al., issued on Jun. 20, 2006, inU.S. Pat. No. 7,217,937 to King, issued on May 15, 2007, in U.S. Pat.No. 7,307,721 to King, issued on Dec. 11, 2007, in U.S. Pat. No.7,379,577 to King, et al., issued on May 27, 2008, and in U.S. Pat. No.7,605,919 to Oma, et al., issued on Oct. 20, 2009, which areincorporated herein by reference. Other examples of bright-field imagingparticle analyzers are described in U.S. Pat. No. 6,061,130 to Plate, etal., issued on May 9, 2000, and in U.S. Pat. No. 6,522,781 to Norikane,et al., issued on Feb. 18, 2003, which are incorporated herein byreference. By using such particle analyzers, particles of an upper sizerange in a liquid sample can be individually analyzed and visualized.

On the other hand, a dark-field imaging particle analyzer may be used todetermine the sizes of particles smaller than about 1 μm in a liquidsample. For example, nanoparticle tracking analysis (NTA) particleanalyzers are described in U.S. Pat. No. 6,280,960 to Carr, issued onAug. 28, 2001, and in U.S. Pat. No. 7,399,600 to Carr, issued on Jul.15, 2008, which are incorporated herein by reference. By using suchparticle analyzers, particles of a lower size range in a liquid samplecan be individually analyzed and visualized. However, as NTA requiresthe capture of several dark-field image frames, the rate of analysis isrelatively low.

Although both bright-field and dark-field imaging particle analyzers areseparately known, the use of different particle analyzers to determinethe sizes of particles of an upper size range and particles of a lowersize range in the same liquid sample is highly inconvenient.Furthermore, the sizes determined by the different particle analyzersoften disagree where the upper and lower size ranges overlap.

An imaging flow cytometer combining bright-field and dark-field imagingis described in U.S. Pat. No. 7,634,125 to Ortyn, et al., issued on Dec.15, 2009, which is incorporated herein by reference. This imaging flowcytometer may be used to determine the sizes of cells in a liquidsample. However, cells of only a narrow size range, typically, about 5μm to 15 μm, can be individually analyzed and visualized.

A particle analyzer combining bright-field imaging and laser-diffractionanalysis is described in U.S. Pat. No. 7,471,393 to Trainer, issued onDec. 30, 2008, which is incorporated herein by reference. This particleanalyzer may be used to determine the sizes of particles of an uppersize range and particles of a lower size range in a liquid sample.However, the particles of the lower size range cannot be individuallyanalyzed or visualized. Rather, the particles of the lower size rangeare analyzed as an array, on the basis of their laser diffractionpattern.

Particle analyzers combining single-particle light-extinction andlight-scattering analysis are described in U.S. Pat. No. 5,835,211 toWells, et al., issued on Nov. 10, 1998, and in U.S. Pat. No. 6,794,671to Nicoli, et al., issued on Sep. 21, 2004, which are incorporatedherein by reference. These particle analyzers may be used to determinethe sizes of particles of an upper size range and particles of a lowersize range in a liquid sample. However, the size determination relies ona calibration curve determined by using external calibration standardsand is prone to calibration errors arising from differences between theoptical properties of the particles and the calibration standards.Moreover, the particles cannot be individually visualized.

Therefore, a particle analyzer combining bright-field and dark-fieldimaging that allows particles of an upper size range and particles of alower size range in a liquid sample to be individually analyzed andvisualized is highly desirable. Such a particle analyzer should provideconsistent sizes throughout the upper and lower size ranges to enable abroad particle size distribution of the liquid sample to be accuratelydetermined.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a method a particle sizedistribution of a liquid sample with a particle analyzer; the liquidsample including particles of a lower size range that are resolvable bydark-field imaging, particles of an intermediate size range that areresolvable by dark-field imaging and by bright-field imaging, andparticles of an upper size range that are resolvable by bright-fieldimaging; the particle analyzer including a sample cell, a dark-fieldlight source, a bright-field light source, an imaging system, and aprocessing system including an analysis module; the method comprising:a) holding the liquid sample in the sample cell; b) illuminating theliquid sample in the sample cell with the dark-field light source in adark-field geometry to yield scattered light; c) collecting, focusing,and detecting the scattered light with the imaging system to capture adark-field image frame in which the particles of the lower size rangeand the particles of the intermediate size range are resolved; d)analyzing the dark-field image frame with the analysis module to locateimages of the particles of the lower size range and the particles of theintermediate size range; e) analyzing the images of the particles of thelower size range and the particles of the intermediate size range withthe analysis module to determine relative sizes of the particles of thelower size range and the particles of the intermediate size range; f)illuminating the liquid sample in the sample cell with the bright-fieldlight source in a bright-field geometry to yield transmitted light; g)collecting, focusing, and detecting the transmitted light with theimaging system to capture a bright-field image frame in which theparticles of the intermediate size range and the particles of the uppersize range are resolved; h) analyzing the bright-field image frame withthe analysis module to locate images of the particles of theintermediate size range and the particles of the upper size range; i)analyzing the images of the particles of the intermediate size range andthe particles of the upper size range with the analysis module todetermine absolute sizes of the particles of the intermediate size rangeand the particles of the upper size range; j) comparing the dark-fieldimage frame and the bright field image frame with the analysis module toidentify corresponding images of the particles of the intermediate sizerange located in both the dark-field image frame and the bright-fieldimage frame; k) comparing the relative sizes and the absolute sizes ofthe particles of the intermediate size range that were determined byanalyzing the corresponding images with the analysis module to determinea calibration curve; l) applying the calibration curve to the relativesizes of the particles of the lower size range with the analysis moduleto determine calibrated sizes of the particles of the lower size range;and m) determining the particle size distribution of the liquid samplefrom the calibrated sizes of the particles of the lower size range, andthe absolute sizes of the particles of the intermediate size range andthe particles of the upper size range with the analysis module.

Another aspect of the present invention relates to a particle analyzerfor determining a particle size distribution of a liquid sample; theliquid sample including particles of a lower size range that areresolvable by dark-field imaging, particles of an intermediate sizerange that are resolvable by dark-field imaging and by bright-fieldimaging, and particles of an upper size range that are resolvable bybright-field imaging; the particle analyzer comprising: a sample cellfor holding the liquid sample; a dark-field light source forilluminating the liquid sample in the sample cell in a dark-fieldgeometry to yield scattered light; a bright-field light source forilluminating the liquid sample in the sample cell in a bright-fieldgeometry to yield transmitted light; an imaging system for collecting,focusing, and detecting the scattered light to capture a dark-fieldimage frame in which the particles of the lower size range and theparticles of the intermediate size range are resolved, and forcollecting, focusing, and detecting the transmitted light to capture abright-field image frame in which the particles of the intermediate sizerange and the particles of the upper size range are resolved; and aprocessing system including an analysis module for analyzing thedark-field image frame to locate images of the particles of the lowersize range and the particles of the intermediate size range, foranalyzing the images of the particles of the lower size range and theparticles of the intermediate size range to determine relative sizes ofthe particles of the lower size range and the particles of theintermediate size range; for analyzing the bright-field image frame tolocate images of the particles of the intermediate size range and theparticles of the upper size range, for analyzing the images of theparticles of the intermediate size range and the particles of the uppersize range to determine absolute sizes of the particles of theintermediate size range and the particles of the upper size range, forcomparing the dark-field image frame and the bright field image frame toidentify corresponding images of the particles of the intermediate sizerange located in both the dark-field image frame and the bright-fieldimage frame, for comparing the relative sizes and the absolute sizes ofthe particles of the intermediate size range that were determined byanalyzing the corresponding images to determine a calibration curve, forapplying the calibration curve to the relative sizes of the particles ofthe lower size range to determine calibrated sizes of the particles ofthe lower size range, and for determining the particle size distributionof the liquid sample from the calibrated sizes of the particles of thelower size range, and the absolute sizes of the particles of theintermediate size range and the particles of the upper size range.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in greater detail with referenceto the accompanying drawings, which represent exemplary embodimentsthereof, wherein:

FIG. 1 is a schematic illustration of a first embodiment of a particleanalyzer according to the present invention;

FIG. 2 is an exemplary dark-field image frame captured by a particleanalyzer according to the present invention;

FIG. 3 is an exemplary bright-field image frame captured by a particleanalyzer according to the present invention;

FIG. 4A is a schematic illustration of a second embodiment of a particleanalyzer according to the present invention;

FIG. 4B is a schematic illustration of a dark-field light source, abright-field light source, a sheet-forming system, and a sample cell ofthe particle analyzer of FIG. 4A; and

FIG. 5 is a schematic illustration of a third embodiment of a particleanalyzer according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method and a particle analyzer fordetermining a broad particle size distribution of a liquid sample byusing a combination of bright-field and dark-field imaging.

Typically, the liquid sample comprises particles of a broad overall sizerange of about 0.05 μm to 500 μm, the particles being dispersed in theliquid sample. The liquid sample comprises particles of a lower sizerange, particles of an intermediate size range, and particles of anupper size range. The particles of the lower size range and theparticles of the intermediate size range are resolvable by dark-fieldimaging, meaning that images of particles of the lower size range andthe particles of the intermediate size range are discernible anddistinguishable in a dark-field image frame. The particles of theintermediate size range and the particles of the upper size range areresolvable by bright-field imaging, meaning that images of particles ofthe intermediate size range and the particles of the upper size rangeare discernible and distinguishable in a bright-field image frame.Preferably, the lower size range is of about 0.05 μm to 0.5 μm, theintermediate size range is of about 0.5 μm to 1 μm, and the upper sizerange is of about 1 μm to 500 μm. Typically, the concentration ofparticles in the liquid sample is less than about 10 000 000particles/mL.

With reference to FIG. 1, a first embodiment of the particle analyzer100 includes a sample cell 110, a dark-field light source 120, abright-field light source 130, an imaging system 140, a processingsystem 150, and a pumping system 160. The imaging system 140 typicallyincludes a magnification system 141 and a detector array 142. Theprocessing system 150 typically includes a control module and ananalysis module (not shown). The bright-field light source 130, thesample cell 110, the magnification system 141, and the detector array142 are aligned along an optical axis 170. The dark-field light source120 and the sample cell 110 are aligned along a direction 171 at anangle 172 to the optical axis 170.

Typically, the sample cell 110, which holds the liquid sample, is a flowcell. The pumping system 160 passes the liquid sample in a flowingstream into the sample cell 110. Preferably, the pumping system 160 is apulse pumping system, as described in U.S. Pat. No. 7,307,721, whichpasses the liquid sample into the sample cell 110 with a pulse. Once theliquid sample is in the sample cell 110, the pumping system 160substantially stops the stream to ensure that the liquid sample issubstantially stationary in the sample cell 110 during image-framecapture. For example, the flow rate may be on the order of about 1 μm/s.After the liquid sample has been imaged, the pumping system 160, withanother pulse, passes the liquid sample in the stream, now flowing onceagain, out of the sample cell 110, while passing a subsequent liquidsample in the stream into the sample cell 110.

The sample cell 110 is positioned to allow both the dark-field lightsource 120 and the bright-field light source 130 to illuminate theliquid sample, and is substantially transparent to light emitted fromthe dark-field light source 120 and the bright-field light source 130.The same optical sampling volume, which may be less than or equal to thevolume of the sample cell 110, is illuminated by the dark-field lightsource 120 and the bright-field light source 130. The dark-field lightsource 120 and the bright-field light source 130 may be lamps,light-emitting diodes, lasers, or any other suitable light sources.

The dark-field light source 120 and the bright-field light source 130may emit light in the same wavelength band or in different wavelengthbands. For instance, when some of the particles in the liquid sample arefluorescent particles, which emit scattered fluorescent light in a firstwavelength band after absorbing light in a second wavelength band, thedark-field light source 120 may be a fluorescent light source thatilluminates the liquid sample in the sample cell 110 with light in thesecond wavelength band, and the bright-field light source 130 mayilluminate the liquid sample in the sample cell 110 with light in thefirst wavelength band. A wavelength selective filter that passes onlylight in the first wavelength band to the detector array 142, whileblocking light in the second wavelength band, may be included in theimaging system 140, as described in further detail hereafter.

The dark-field light source 120 illuminates the liquid sample in adark-field geometry to yield scattered light. In other words, thedark-field light source 120 laterally illuminates the liquid sample toproduce an image from light scattered by the liquid sample, in whichparticles appear bright against a dark background. The dark-field lightsource 120 illuminates the liquid sample along a direction 171 at anangle 172 to the optical axis 170 to ensure that a suitable scatteredsignal from the liquid sample is received by the imaging system 140,while minimizing unwanted scatter from the sample cell 110. The angle172 may be any angle.

The bright-field light source 130 illuminates the liquid sample in abright-field geometry to yield transmitted light. In other words, thebright-field light source 130 trans-illuminates the liquid sample toproduce an image from light transmitted by the liquid sample, in whichparticles appear dark against a bright background. The bright-fieldlight source 130 illuminates the liquid sample along the optical axis170 to ensure that a suitable transmitted signal from the liquid sampleis received by the imaging system 140.

The dark-field light source 120 and the bright-field light source 130are separately activated, typically in alternation, so that either thescattered light yielded by the illumination of the liquid sample withthe dark-field light source 120 or the transmitted light yielded by theillumination of the liquid sample with the bright-field light source 130is received by the imaging system 140. Typically, the liquid sample isilluminated once with the dark-field light source 120 and once with thebright-field light source 130 to acquire a set of two image frames.

The magnification system 141 of the imaging system 140 collects thescattered light or the transmitted light and focuses it onto thedetector array 142 of the imaging system 140. Typically, themagnification system 141 includes an objective, which may be anobjective lens, a set of objective lenses of different magnifications, avariable-magnification lens system, or any other suitable objective, aswell as one or more tube lenses. Preferably, the magnification system141 also includes a diaphragm for enhancing diffraction enlargement, asdescribed in U.S. Pat. No. 7,379,577.

The magnification system 141 of the imaging system 140 has amagnification factor and a numerical aperture selected to provide asuitable field of view and a suitable depth of field. The field of viewof the magnification system 141, which corresponds to thecross-sectional area of the optical sampling volume, and the depth offield of the magnification system 141, which corresponds to the depth ofthe optical sampling volume, decrease as the magnification factor andthe numerical aperture increase. The depth of field is usually small.For instance, the magnification system may have a magnification factorof 5, a field of view of about 2.2 mm², and a depth of field of about100 μm, or a magnification factor of 10, a field of view of about 0.5mm², and a depth of field of about 30 μm. Preferably, the liquid sampleis confined to the maximum depth of field of the magnification system141. For instance, the sample cell 110 may be designed to have a depththat is less than or equal to the maximum depth of field of themagnification system 141.

The detector array 142 of the imaging system 140 detects the scatteredlight or the transmitted light to capture a dark-field or bright-fieldimage frame, respectively. The detector array 142 includes a pluralityof detector elements. Typically, the detector array includes greaterthan 1 000 000 detector elements of about 4 μm to 6 μm in size. Thedetector array 142 detects the intensity of light incident on theplurality or detector elements and thereby captures a digital imageframe formed of a plurality of pixels. The detector array 142 may be acharge-coupled device (CCD) array, a complementary metal-oxidesemiconductor (CMOS) array, or any other suitable detector array.

Although it is preferred that a single detector array 142 of the imagingsystem 140 detects both the scattered light and the transmitted light,other embodiments may include two separate detector arrays 142 fordetecting the scattered light and the transmitted light, respectively.For instance, in such an embodiment, the dark-field light source 120 andthe bright-field light source 130 may emit light in two differentwavelength bands, and two wavelength selective filters that each passonly light in one of the wavelength bands to a respective detector array142, while blocking light in the other wavelength band, may be includedin the imaging system 140.

A set of image frames, typically consisting of a dark-field image frameand a bright-field image frame, is captured for each liquid sample. Inoperation, the pumping system 160 passes the liquid sample in a flowingstream into the sample cell 110 and then substantially stops the stream.The dark-field light source 120 illuminates the liquid sample in thesample cell 110, and the detector array 142 of the imaging system 140captures a dark-field image frame. The bright-field light source 130illuminates the liquid sample in the sample cell 110, and the detectorarray 142 captures a bright-field image frame. The pumping system 160then passes the liquid sample in the stream out of the sample cell 110,while passing a subsequent liquid sample in the stream, now flowing onceagain, into the sample cell 110. These steps are repeated until therequired volume of liquid has been analyzed.

The time and intensity of illumination provided by the dark-field lightsource 120 and the bright-field light source 130 during image-framecapture, the flow rate of the liquid sample during image-frame captureas regulated by the pumping system 160, and the rate of image-framecapture by the detector array 142 of the imaging system 140 are selectedto ensure that “freeze frame” conditions prevail as the set of imageframes is captured, meaning that particles move by less than asignificant fraction, typically about 5% to 15%, of their dimensions.

The control module of the processing system 150 controls the detectorarray 142 to determine the rate of image-frame capture. Preferably, thecontrol module also controls the dark-field light source 120 and thebright-field light source 130 to determine the time of illumination, aswell as the pumping system 160 to determine the flow rate. Thereby, thecontrol module synchronizes the dark-field light source 120, thebright-field light source 130, the pumping system 160, and the detectorarray 142 of the imaging system 140 to ensure that the dark-field imageframe and the bright-field image frame are successively captured, ineither order, while the liquid sample is substantially stationary in thesample cell 110.

The processing system 150 receives the captured dark-field andbright-field image frames from the detector array 142 of the imagingsystem 140, stores the image frames, displays the image frames forviewing, and analyzes the image frames. Typically, the processing system150 includes a memory and a suitably programmed processor, such as acentral processing unit (CPU), a digital signal processor (DSP), afield-programmable gate array (FPGA), or any other suitable processor.The control module and the analysis module of the processing system 150are typically implemented as software.

The analysis module of the processing system 150 records backgroundintensities of each pixel when no sample particles are present in thesample cell 110. These background intensities are used to performbackground subtraction and to minimize the effects of stuck particles orother fixed artifacts in the sample cell 110.

In the dark-field image frame, the particles of the lower size range andthe particles of the intermediate size range are resolved. Generally,the particles of the upper size range are not resolved in the dark-fieldimage frame because of blooming effects. Scattered particle imagesappear in the dark-field image frame as bright “stars” against a darkbackground. An exemplary dark-field image frame 243 is shown in FIG. 2.

The analysis module of the processing system 150 first analyzes thedark-field image frame to locate the images of the particles of thelower size range and the particles of the intermediate size range. Theanalysis module compares the intensity of each pixel in the digitalimage frame to a predetermined intensity threshold. The predeterminedintensity threshold is selected to provide the most sensitive detectionof pixels located wholly or partially in particle images, whileminimizing incorrect counting of pixels whose intensity varies becauseof optical and/or electrical noise. If a cluster of adjacent pixels,typically at least 5 adjacent pixels, have intensities larger than theintensity threshold, the cluster is interpreted as a particle image, andthe location of the particle image in the dark-field image frame isstored.

The analysis module of the processing system 150 then analyzes thelocated images of the particles of the lower size range and theparticles of the intermediate size range by determining their sizes andintensities. The size of each particle image is determined by countingthe pixels within the particle image. The intensity of each particleimage is determined by averaging the intensities of the pixels withinthe particle image. The size and intensity of the particle image aredependent on the scattering power of that particle. The scattering powerof the particle, in turn, is dependent on the size of the particle, aswell as factors such as the optical properties of the particle, and theangles of illumination and detection. On this basis, the analysis moduledetermines the relative sizes of the particles of the lower size rangeand the particles of the intermediate size range from the sizes andintensities of their particle images. However, as the scattering powerof a particle increases rapidly with size, leading to oversaturation ofthe detector elements and blooming effects in the particle image, therelative sizes of the particles of the upper size range cannot bedetermined reliably.

In the bright-field image frame, the particles of the intermediate sizerange and the particles of the upper size range are resolved. Generally,the particles of the lower size range are not resolved in thebright-field image frame because of insufficient contrast. Transmittedparticle images appear in the bright-field image frame as dark shadowsagainst a bright background. An exemplary bright-field image frame 344is shown in FIG. 3.

The analysis module of the processing system 150 first analyzes thebright-field image frame to locate the images of the particles of theintermediate size range and the particles of the upper size range. Theanalysis module compares the intensity of each pixel in the digitalimage frame to a predetermined intensity threshold. The predeterminedintensity threshold is selected to provide the most sensitive detectionof pixels located wholly or partially in particle images, whileminimizing incorrect counting of pixels whose intensity varies becauseof optical and/or electrical noise. If a cluster of adjacent pixels,typically at least 5 adjacent pixels, have intensities smaller than theintensity threshold, the cluster is interpreted as a particle image, andthe location of the particle image in the bright-field image frame isstored.

The analysis module of the processing system 150 then analyzes thelocated images of the particles of the intermediate size range and theparticles of the upper size range by determining their sizes. The sizeof each particle image is determined by counting the number of pixelswithin the particle image. The size of the particle image is related tothe size of that particle by the magnification factor of themagnification system 141 and any additional diffraction enlargement.Advantageously, the size of the particle image is substantiallyindependent of factors such as the optical properties of the particle.On this basis, the analysis module determines the absolute sizes of theparticles of the intermediate size range and the particles of the uppersize from the sizes of their particle images. However, as theinteraction of a particle with the bright-field illumination decreaseswith size, leading to insufficient contrast between the particle imageand the background, and to an insufficient number of pixels in theparticle image, absolute sizes of the particles of the lower size rangecannot be determined reliably. In some instances, it may be desirable touse contrast enhancement techniques, as described in U.S. Pat. No.7,605,919.

The analysis module of the processing system 150 compares the dark-fieldimage frame and the bright-field image frame to identify correspondingimages of the particles of the intermediate size located in both thedark-field image frame and the bright-field image frame. For eachparticle image in the dark-field image frame, the analysis modulecompares its stored location with the stored locations of the particleimages in the bright-field image frame. If the stored locations of a setof particle images differ by less than a predetermined displacementthreshold, the set of particle images are interpreted as correspondingimages of the same particle of the intermediate size range. For example,the predetermined displacement threshold may be less than about 5% ofthe length of the field of view and less than about 1% of the width ofthe field of view.

In effect, the particles of the intermediate size range, for whichcorresponding particle images are located in the dark-field and thebright-field image frames, serve as internal calibration standards. Theanalysis module of the processing system 150 determines a calibrationcurve by comparing the relative sizes and the absolute sizes of theparticles of the intermediate size range, which were determined byanalyzing the corresponding images of the particles of the intermediatesize range located in the dark-field image frame and the bright-fieldimage frame, respectively. Generally, the calibration curve is generatedby fitting the size data, for example, by using a polynomial function.Typically, a look-up table is also generated.

The analysis module of the processing system 150 then applies thecalibration curve to the relative sizes of the particles of the lowersize range to determine calibrated sizes of the particles of the lowersize range. In other words, the analysis module uses the calibrationcurve to convert the relative sizes of the particles of the lower sizerange, which were determined by analyzing the images of the particles ofthe lower size range located in the dark-field image frame, tocalibrated sizes.

Finally, the analysis module of the processing system 150 determines theparticle size distribution of the liquid sample from the calibratedsizes of the particles of the lower size range, and the absolute sizesof the particles of the intermediate size range and the particles of theupper size range. Typically, the analysis module of the processingsystem 150 also displays the particle size distribution for viewing.

With reference to FIGS. 4A and 4B, a second embodiment of the particleanalyzer 400 is similar to the first embodiment, but includes aspecially adapted sample cell 410, and a dark-field light source 420that is aligned with the sample cell 410 along a direction 471 at anangle 472 of about 90° to the optical axis 170.

The specially adapted sample cell 410 includes a side window 411 that issubstantially transparent to light emitted from the dark-field lightsource 420. The sample cell 410 also includes a front window 412 and aback window 413 that are substantially transparent to light emitted fromthe bright-field light source 130. Preferably, the front window 412 andthe back window 413 are substantially parallel and are separated by adepth that is less than or equal to the maximum depth of field of themagnification system 141. For example, the sample cell 110 may have adepth of about 100 μm or about 30 μm.

The sample cell 410 is positioned such that the side window 411 receiveslight from the dark-field light source 420 and the front window 412receives light from the bright-field light source 130. The dark-fieldlight source 420 illuminates the liquid sample along the direction 471substantially orthogonal to the optical axis 170, and as in the firstembodiment, the bright-field light source 130 illuminates the liquidsample along the optical axis 170.

With particular reference to FIG. 4B, preferably, the dark-field lightsource 420 is a laser, and the particle analyzer 400 includes asheet-forming system 421, which is positioned between the dark-fieldlight source 420 and the sample cell 410. The sheet-forming system 421forms the light emitted from the dark-field light source 420 into alight sheet substantially parallel to the front window 412 and the backwindow 413, and directed into the side window 411 of the sample cell410.

In one embodiment, the sheet-forming system 421 includes a sphericallens 422 and a cylindrical lens 423. The spherical lens 422 collimatesthe light emitted from the dark-field light source 420, and thecylindrical lens 423 focuses the collimated light into a light sheetdirected into the side window 411 of the sample cell 410. Any othersuitable embodiment of a sheet-forming system may also be used.

Typically, the focused light sheet, in free space, has a Gaussianintensity distribution, and the intensity of the light sheet variesalong its direction of propagation and along its narrow direction. Theseintensity variations can reduce the resolution with which particles ofdifferent sizes can be distinguished.

Advantageously, the sample cell 410 reduces such intensity variations inthe light sheet by effectively serving as an optical waveguide. Thefront window 412 and the back window 413 of the sample cell 410 serve ashighly reflective, substantially parallel walls, separated by a narrowdepth. The high degree of reflection results from the low angle ofincidence of the light sheet on the front window 412 and the back window413. The reflection is further enhanced by ensuring that thepolarization axis of the light sheet lies in a plane substantiallyparallel to the front window 412 and the back window 413. Whenpropagating through the sample cell 410, the light sheet is partiallyconfined and diverges to a lesser degree than it would in free space.The partial confinement also reduces the angular accuracy with which thelight sheet must be directed between the front window 412 and the backwindow 413. Preferably, the front window 412 and the back window 413 areformed of a low-index glass, such as silica.

With reference to FIG. 5, a third embodiment of the particle analyzer500 is useful in instances where some of the particles in the liquidsample are fluorescent particles, having either natural fluorophores orfluorophore tags, which emit fluorescent light in a first wavelengthband after absorbing light in a second wavelength band.

The third embodiment of the particle analyzer 500 is similar to thefirst embodiment, but includes an additional fluorescence light source580 that is aligned with the sample cell 110 along a direction 573 at anangle 574 to the optical axis 170, and an imaging system 540 comprisinga wavelength selective filter 543. The angle 574 may be any angle.

The fluorescence light source 580 illuminates the liquid sample in thesample cell 110 with light in the second wavelength band to yieldfluorescent light in the first wavelength band, as well as scatteredlight in the second wavelength band. The dark-field light source 120 andthe bright-field light source 130 illuminate the liquid sample in thesample cell 110 with light in the first wavelength band to yieldscattered light or transmitted light, respectively, in the firstwavelength band. Typically, the liquid sample is illuminated once withthe dark-field light source 120, once with the bright-field light source130, and once with the fluorescence light source 580 to capture a set ofthree image frames, consisting of a dark-field image frame, abright-field image frame, and a fluorescence image frame. The dark-fieldimage frame, the bright-field image frame, and the fluorescence imageframe are successively captured, in any order, while the liquid sampleis substantially stationary in the sample cell 110, that is, under“freeze frame” conditions, as described heretofore.

The wavelength selective filter 543 of the imaging system 540 passes thescattered light, the transmitted light, and the fluorescent light in thefirst wavelength band to the detector array 142 of the imaging system540, while blocking the scattered light in the second wavelength band.The wavelength selective filter 543 is preferably positioned within themagnification system 141 of the imaging system 540, but may also bepositioned between the magnification system 141 and the detector array142 or between the sample cell 110 and the magnification system 141.

Accordingly, the magnification system 141 of the imaging system 540collects and focuses the scattered light, the transmitted light, or thefluorescent light in the first wavelength band onto the detector array142 of the imaging system 540. The detector array 142 detects thescattered light, the transmitted light, or the fluorescent light in thefirst wavelength band to capture a dark-field, bright-field, orfluorescence image frame, respectively.

The processing system 150 receives the captured dark-field,bright-field, and fluorescence image frames from the detector array 142,stores the image frames, displays the image frames for viewing, andanalyzes the image frames. The dark-field and bright-field image framesare analyzed as described heretofore.

The analysis module of the processing system 150 first analyzes thefluorescence image frame to locate images of the fluorescent particles.The analysis module compares the intensity of each pixel in the digitalimage frame to a predetermined intensity threshold. The predeterminedintensity threshold is selected to provide the most sensitive detectionof pixels located wholly or partially in particle images, whileminimizing incorrect counting of pixels whose intensity varies becauseof optical and/or electrical noise. If a cluster of adjacent pixels,typically at least 5 adjacent pixels, have intensities larger than theintensity threshold, the cluster is interpreted as a particle image, andthe location of the particle image in the fluorescence image frame isstored.

The analysis module of the processing system 150 then compares thedark-field, bright-field, and fluorescence image frames to identifycorresponding images of the fluorescent particles in the fluorescenceimage frame and in either or both of the dark-field and bright-fieldimage frames. For each particle image in the fluorescence image frame,the analysis module compares its stored location with the storedlocations of the particle images in the dark-field and bright-fieldimage frames. If the stored locations of a set of particle images differby less than a predetermined displacement threshold, the set of particleimages are interpreted as corresponding images of the same fluorescentparticle. For example, the predetermined displacement threshold may beless than about 5% of the length of the field of view and less thanabout 1% of the width of the field of view.

Thereby, the analysis module of the processing system 150 may identifyimages of the fluorescent particles of the lower size range and thefluorescent particles of the intermediate size range in the dark-fieldimage frame, and may identify images of the fluorescent particles of theintermediate size range and the fluorescent particles of the upper sizerange in the bright-field image frame. Furthermore, the analysis modulemay determine a size distribution of only the fluorescent particles inthe liquid sample from the calibrated sizes of the fluorescent particlesof the lower size range, and the absolute sizes of the fluorescentparticles of the intermediate size range and the fluorescent particlesof the upper size range.

Of course, numerous other embodiments may be envisaged without departingfrom the spirit and scope of the invention.

We claim:
 1. A method, comprising: a) holding a liquid sample in asample cell of a particle analyzer, the liquid sample includingparticles of a lower size range that are resolvable by dark-fieldimaging, particles of an intermediate size range that are resolvable bydark-field imaging and by bright-field imaging, and particles of anupper size range that are resolvable by bright-field imaging, some ofthe particles in the liquid sample being fluorescent particles that emitlight in a first wavelength band after absorbing light in a secondwavelength band, wherein the particle analyzer includes the sample cell,a dark-field light source, a bright-field light source, a fluorescencelight source, a processing system, and an imaging system including amagnification sub-system, a detector array and a wavelength selectivefilter; b) illuminating the liquid sample in the sample cell with lightin the first wavelength band with the dark-field light source in adark-field geometry to yield scattered light in the first wavelengthband; c) collecting, focusing, and detecting the scattered light in thefirst wavelength band with the imaging system to capture a dark-fieldimage frame in which the particles of the lower size range and theparticles of the intermediate size range are resolved, wherein thescattered light in the first wavelength band is collected and focusedonto the detector array of the imaging system with the magnificationsub-system, and is detected with the detector array to capture thedark-field image frame; d) analyzing the dark-field image frame with ananalysis module of the processing system to locate images of theparticles of the lower size range and the particles of the intermediatesize range; e) analyzing the images of the particles of the lower sizerange and the particles of the intermediate size range from thedark-field image frame with the analysis module of the processing systemto determine relative sizes of the particles of the lower size range andthe particles of the intermediate size range; f) illuminating the liquidsample in the sample cell with light in the first wavelength band withthe bright-field light source in a bright-field geometry to yieldtransmitted light in the first wavelength band; g) collecting, focusing,and detecting the transmitted light in the first wavelength band withthe imaging system to capture a bright-field image frame in which theparticles of the intermediate size range and the particles of the uppersize range are resolved, wherein the transmitted light in the firstwavelength band is collected and focused onto the detector array of theimaging system with the magnification sub-system, and is detected withthe detector array to capture the bright-field image frame; h) analyzingthe bright-field image frame with the analysis module of the processingsystem to locate images of the particles of the intermediate size rangeand the particles of the upper size range; i) analyzing the images ofthe particles of the intermediate size range and the particles of theupper size range from the bright-field image frame with the analysismodule of the processing system to determine absolute sizes of theparticles of the intermediate size range and the particles of the uppersize range; j) comparing the dark-field image frame and the bright fieldimage frame with the analysis module of the processing system toidentify corresponding images of the particles of the intermediate sizerange located in both the dark-field image frame and the bright-fieldimage frame; k) comparing the relative sizes and the absolute sizes ofthe particles of the intermediate size range that were determined byanalyzing the corresponding images with the analysis module of theprocessing system to determine a calibration curve; l) applying thecalibration curve to the relative sizes of the particles of the lowersize range with the analysis module of the processing system todetermine calibrated sizes of the particles of the lower size range; m)illuminating the liquid sample in the sample cell with light in thesecond wavelength band with the fluorescence light source to yieldfluorescent light in the first wavelength band and scattered light inthe second wavelength band; n) passing the scattered light, thetransmitted light, and the fluorescent light in the first wavelengthband to the detector array with the wavelength selective filter of theimaging system, while blocking the scattered light in the secondwavelength band with the wavelength selective filter; o) collecting andfocusing the fluorescent light in the first wavelength band onto thedetector array with the magnification sub-system; p) detecting thefluorescent light in the first wavelength band with the detector arrayto capture a fluorescence image frame; q) analyzing the fluorescenceimage frame with the analysis module of the processing system to locateimages of the fluorescent particles; and r) comparing the fluorescenceimage frame, the dark-field image frame, and the bright-field imageframe with the analysis module of the processing system to identifycorresponding images of the fluorescent particles located in thefluorescence image frame and in either or both of the dark-field imageframe and the bright-field image frame; s) determining a particle sizedistribution of the liquid sample from the calibrated sizes of thefluorescent particles of the lower size range, and the absolute sizes ofthe fluorescent particles of the intermediate size range and thefluorescent particles of the upper size range with the analysis moduleof the processing system.
 2. The method of claim 1, wherein the lowersize range is of about 0.05 μm to 0.5 μm, the intermediate size range isof about 0.5 μm to 1 μm, and the upper size range is of about 1 μm to500 μm.
 3. The method of claim 1, wherein the particle analyzer furtherincludes a pumping system, the method further comprising, prior to a):passing the liquid sample in a flowing stream into the sample cell withthe pumping system; and substantially stopping the flowing stream withthe pumping system when the liquid sample is in the sample cell toensure that the liquid sample is substantially stationary in the samplecell.
 4. The method of claim 3, wherein the processing system furtherincludes a control module, the method further comprising: synchronizingthe dark-field light source, the bright-field light source, the detectorarray, and the pumping system with the control module to ensure that thedark-field image frame and the bright-field image frame are successivelycaptured while the liquid sample is substantially stationary in thesample cell.
 5. The method of claim 1, wherein the bright-field lightsource, the sample cell, the magnification sub-system, and the detectorarray are aligned along an optical axis; wherein the dark-field lightsource and the sample cell are aligned along a direction at an angle tothe optical axis; wherein b) includes illuminating the liquid sample inthe sample cell along the direction at an angle to the optical axis withthe dark-field light source; and wherein f) includes illuminating theliquid sample in the sample cell along the optical axis with thebright-field light source.
 6. The method of claim 5, wherein thedark-field light source and the sample cell are aligned along adirection at an angle of about 90 degrees to the optical axis; andwherein b) includes illuminating the liquid sample in the sample alongthe direction at an angle of about 90 degrees to the optical axis. 7.The method of claim 6, wherein the particle analyzer further includes asheet-forming system; wherein the sample cell includes a side window forreceiving light emitted from the dark-field light source, a front windowfor receiving light emitted from the bright-field light source, and aback window substantially parallel to the front window and separatedtherefrom by a depth that is less than or equal to a maximum depth offield of the magnification sub-system; wherein the dark-field lightsource is a laser; and wherein b) further includes forming the lightemitted from the dark-field light source into a light sheetsubstantially parallel to the front window and the back window of thesample cell, and directed into the side window of the sample cell withthe sheet-forming system.
 8. An apparatus, comprising: a particleanalyzer configured to determine a particle size distribution of aliquid sample, the liquid sample including particles of a lower sizerange that are resolvable by dark-field imaging, particles of anintermediate size range that are resolvable by dark-field imaging and bybright-field imaging, and particles of an upper size range that areresolvable by bright-field imaging, some of the particles in the liquidsample being fluorescent particles that emit light in a first wavelengthband after absorbing light in a second wavelength band, the particleanalyzer including: a sample cell configured to holding the liquidsample; a dark-field light source configured to illuminate the liquidsample in the sample cell with light in the first wavelength band in adark-field geometry to yield scattered light in the first wavelengthband; a bright-field light source configured to illuminate the liquidsample in the sample cell with light in the first wavelength band in abright-field geometry to yield transmitted light in the first wavelengthband; a fluorescence light source configured to illuminate the liquidsample in the sample cell with light in the second wavelength band toyield fluorescent light in the first wavelength band and scattered lightin the second wavelength band; an imaging system including a wavelengthselective filter, a detector array and a magnification sub-system, thewavelength selective filter configured to pass the scattered light, thetransmitted light, and the fluorescent light in the first wavelengthband to the detector array while blocking the scattered light in thesecond wavelength band, the magnification sub-system configured tocollect and focus the scattered light, the transmitted light, and thefluorescent light in the first wavelength band onto the detector array,the detector array configured to detect the scattered light in the firstwavelength band to capture a dark-field image frame in which theparticles of the lower size range and the particles of the intermediatesize range are resolved, the detector array configured to detect thetransmitted light in the first wavelength band to capture a bright-fieldimage frame in which the particles of the intermediate size range andthe particles of the upper size range are resolved, the detector arrayconfigured to detect the fluorescent light in the first wavelength bandto capture a fluorescence image frame; and a processing system includingan analysis module configured to analyze the dark-field image frame tolocate images of the particles of the lower size range and the particlesof the intermediate size range, the analysis module configured toanalyze the images of the particles of the lower size range and theparticles of the intermediate size range from the dark-field image frameto determine relative sizes of the particles of the lower size range andthe particles of the intermediate size range, the analysis moduleconfigured to analyze the bright-field image frame to locate images ofthe particles of the intermediate size range and the particles of theupper size range, the analysis module configured to analyze the imagesof the particles of the intermediate size range and the particles of theupper size range from the bright-field image frame to determine absolutesizes of the particles of the intermediate size range and the particlesof the upper size range, the analysis module configured to analyze thefluorescence image frame to locate images of the fluorescent particles,the analysis module configured to compare the fluorescence image frame,the dark-field image frame, and the bright-field image frame to identifycorresponding images of the fluorescent particles located in thefluorescence image frame and in at least one of the dark-field imageframe or the bright-field image frame, the analysis module configured tocompare the dark-field image frame and the bright field image frame toidentify corresponding images of the particles of the intermediate sizerange located in both the dark-field image frame and the bright-fieldimage frame, the analysis module configured to compare the relativesizes and the absolute sizes of the particles of the intermediate sizerange that were determined by analyzing the corresponding images todetermine a calibration curve, the analysis module configured to applythe calibration curve to the relative sizes of the particles of thelower size range to determine calibrated sizes of the particles of thelower size range, the analysis module configured to determine theparticle size distribution of the liquid sample from the calibratedsizes of the fluorescent particles of the lower size range, and theabsolute sizes of the fluorescent particles of the intermediate sizerange and the fluorescent particles of the upper size range.
 9. Theapparatus of claim 8, wherein the lower size range is of about 0.05 μmto 0.5 μm, the intermediate size range is of about 0.5 μm to 1 μm, andthe upper size range is of about 1 μm to 500 μm.
 10. The apparatus ofclaim 8, wherein the detector array includes a charge-coupled device(CCD) array; and wherein the magnification sub-system includes anobjective, one or more tube lenses, and a diaphragm.
 11. The apparatusof claim 8, further comprising: a pumping system for passing the liquidsample in a flowing stream into the sample cell, and for substantiallystopping the flowing stream when the liquid sample is in the sample cellto ensure that the liquid sample is substantially stationary in thesample cell.
 12. The apparatus of claim 11, wherein the processingsystem further includes a control module configured to synchronize thedark-field light source, the bright-field light source, the detectorarray, and the pumping system to ensure that the dark-field image frameand the bright-field image frame are successively captured while theliquid sample is substantially stationary in the sample cell.
 13. Theapparatus of claim 8, wherein the bright-field light source, the samplecell, the magnification sub-system, and the detector array are alignedalong an optical axis; wherein the dark-field light source and thesample cell are aligned along a direction at an angle to the opticalaxis; wherein the dark-field light source is configured to illuminatethe liquid sample in the sample cell along the direction at an angle tothe optical axis; and wherein the bright-field light source isconfigured to illuminate the liquid sample in the sample cell along theoptical axis.
 14. The apparatus of claim 13, wherein the dark-fieldlight source and the sample cell are aligned along a direction at anangle of about 90 degrees to the optical axis; and wherein thedark-field light source is configured to illuminate the liquid sample inthe sample cell along the direction at an angle of about 90 degrees tothe optical axis.
 15. The apparatus of claim 14, wherein the sample cellincludes a side window configured to receive light emitted from thedark-field light source, a front window configured to receive lightemitted from the bright-field light source, and a back windowsubstantially parallel to the front window and separated therefrom by adepth that is less than or equal to a maximum depth of field of themagnification sub-system.
 16. The apparatus of claim 15, furthercomprising: a sheet-forming system configured to form the light emittedfrom the dark-field light source into a light sheet substantiallyparallel to the front window and the back window of the sample cell, anddirected into the side window of the sample cell; wherein the dark-fieldlight source is a laser.